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US20150299412A1 - Thiol Acrylate Nanocomposite Foams - Google Patents

Thiol Acrylate Nanocomposite Foams Download PDF

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US20150299412A1
US20150299412A1 US14/439,856 US201314439856A US2015299412A1 US 20150299412 A1 US20150299412 A1 US 20150299412A1 US 201314439856 A US201314439856 A US 201314439856A US 2015299412 A1 US2015299412 A1 US 2015299412A1
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thiol
acrylate
copolymer
peta
groups
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Daniel Hayes
John Pojman
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Louisiana State University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/04—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by a previously added blowing agent
    • C08J9/12—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by a previously added blowing agent by a physical blowing agent
    • C08J9/122—Hydrogen, oxygen, CO2, nitrogen or noble gases
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/10—Esters
    • C08F222/1006—Esters of polyhydric alcohols or polyhydric phenols
    • C08F222/103—Esters of polyhydric alcohols or polyhydric phenols of trialcohols, e.g. trimethylolpropane tri(meth)acrylate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
    • C08G63/688—Polyesters containing atoms other than carbon, hydrogen and oxygen containing sulfur
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/0066—Use of inorganic compounding ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/112—Phosphorus-containing compounds, e.g. phosphates, phosphonates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2381/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing sulfur with or without nitrogen, oxygen, or carbon only; Polysulfones; Derivatives of such polymers

Definitions

  • This invention pertains to thiol-acrylate copolymers, thiol-alkyne copolymers, copolymer/ceramic composites, and their use as biocompatible and absorbable materials for tissue repair and regeneration, particularly bone repair and regeneration.
  • Bone grafts have been the standard treatment to augment or accelerate bone regeneration for decades. Autogenous cancellous bone grafts have been used to facilitate bone regrowth, although quantity is limited and surgical procedures for graft harvest are required. Allogeneic bone grafts are costly, require time-consuming bone banking procedures, have high potential for disease transmission, and can result in host-versus-graft disease. Neither technique provides a clinically convenient method for conformal filling of a critical sized bone defect, i.e., a defect whose size is such that the defect will not readily heal spontaneously.
  • Engineered synthetic bone tissue has emerged as an alternative to allogeneic or autogenic bone grafts. This field will become an important alternative to current surgical techniques to replace or restore the function of traumatized, damaged, or lost bone. Desirable features of an engineered composite bone scaffold include a biodegradable composition, broad biocompatibility, large pore volume, and osteoinductive/conductive properties.
  • Native bone contains hydroxyapatite crystals (HA) distributed within an organic matrix.
  • HA hydroxyapatite crystals
  • the porosity and degree of mineralization vary among different bone types.
  • bone scaffold substrates including various natural products, gelatin/bioactive ceramics, and poly( ⁇ -hydroxyesters).
  • gelatin/bioactive ceramics such as calcium and magnesium phosphates, have been studied as well.
  • Synthetic hydroxyapatite composites with degradable polymers such as poly-L-lactic acid, (PLLA) polyglycolic acid (PGA), or poly- ⁇ -caprolactone (PCL), have been used in hybrid cell/scaffold constructs. Scaffold constructs combined with mesenchymal stromal/stem cells (MSC) have resulted in improved bone formation.
  • degradable polymers such as poly-L-lactic acid, (PLLA) polyglycolic acid (PGA), or poly- ⁇ -caprolactone (PCL)
  • the fabrication method itself can have a substantial impact on the mechanical properties and the function of the scaffold, affecting pore size, pore volume, and void interconnectivity. In turn, these factors influence cell attachment, proliferation, extracellular matrix production, and the transport of nutrients and wastes.
  • Common approaches include solid freeform fabrication, thermal precipitation, gas foaming, and solvent casting (often followed by particulate leaching).
  • Thiol-ene chemistries such as thiol-acrylate chemistries have been used in tissue engineering applications. When a thiol-acrylate chemistry is used, all or nearly all of the materials used in the synthesis are incorporated into the complete, finished network, thus reducing the risks of leaching toxic monomers or short chain oligomers. Additionally, bioactive compounds such as peptides, proteins, enzymes, oligonucleotides, and nucleic acids can be copolymerized with thiol-acrylate chemistries. Previous thiol-acrylate chemistries used in biomedical applications have been photolytically polymerized using radical-based photoinitiators.
  • novel thiol-acrylate copolymers that are useful, for example, as injectable biomaterials to provide both mechanical support and biological cues to stimulate bone regrowth.
  • the novel composite is gas-foamed with a blowing agent during or before cure to form a porous, interconnected scaffold.
  • the novel synthesis employs an amine-catalyzed Michael addition co-polymerization of a poly-thiol with a poly-acrylate.
  • the catalyst is an in situ catalyst, such as a tertiary amine moiety that is covalently bonded to one of the reactants, preferably to the poly-acrylate.
  • the materials can rapidly co-polymerize in vivo or in vitro via catalysis by the “attached” in situ tertiary amine groups.
  • FIGS. 1A through 1D One embodiment of the novel synthesis is depicted in FIGS. 1A through 1D .
  • the reagents in the novel synthesis are: (1) a poly-acrylate containing two or more acrylate groups, preferably a tri-acrylate; (2) a poly-thiol containing two or more thiol groups, preferably a tri-thiol; and (3) ammonia, a primary amine, a secondary amine, or a tertiary amine, preferably a secondary amine.
  • At least tri-functionality in the thiol or the acrylate is needed for crosslinking.
  • both the thiol and the acrylate are trifunctional.
  • Using ammonia or a primary amine rather than a secondary amine in the initial step of forming the in situ catalyst also enhances crosslinking, as it effectively increases the number of acrylate groups in those molecules that contain an amine center after the in situ tertiary amine catalyst is formed.
  • FIGS. 1A through 1D The steps of one embodiment of the in situ amine-catalyzed, anionic step growth polymerization mechanism are illustrated in FIGS. 1A through 1D .
  • the first step, depicted in FIG. 1A is the formation of an in situ catalyst/comonomer molecule through the Michael addition of a secondary amine across the carbon-carbon double bond of one of the acrylate groups in the tri-acrylate monomer.
  • the rate of the later polymerization steps can be tuned by adjusting the percentage of acrylate groups that react with an amine functionality. In prototype embodiments to date, this percentage has typically been on the order of ⁇ 1%, but the percentage may be adjusted up or down to fit particular needs. This percentage is preferably between about 0.1% and about 10%.
  • 1A is a secondary amine.
  • the resulting secondary or primary amine product can continue to react with other thiol groups until a tertiary amine-thiol product results, thereby increasing the level of cross-linking (if enhanced cross-linking is desired).
  • the second step, depicted in FIG. 1B is to react the “activated” acrylates (i.e., those incorporating an in situ amine catalytic group) with a thiol comonomer (e.g., TMPTMP as depicted in FIG. 1B ).
  • the amine abstracts a hydrogen from a thiol group, producing a thiol anion.
  • reaction then proceeds via an anionic chain reaction mechanism with a sequential chain transfer step after each addition.
  • An alternative propagation route is depicted in FIG. 1D .
  • the in situ catalyst is a tertiary amine
  • the novel synthesis has no possibility for leaching potentially toxic free radical initiators, as would be the case with a more typical radical polymerization mechanism.
  • Unreacted monomer and unreacted amine may remain in low concentrations embedded within the polymer product, but they would not be expected to be especially reactive once chain propagation has ended; and furthermore their molecular weights are typically such that they would tend to diffuse from within the polymer product only slowly, and therefore they should not present a substantial risk in vivo.
  • this chemistry is more attractive for in situ polymerization in a bone defect than are comparable free radical-based methods.
  • novel copolymer products themselves differ from copolymers that could be synthesized by free radical reaction, in that there is more control over the nature and degree of cross-linking.
  • free-radical polymerization it is not possible to create a true 1:1 copolymer of a thiol and an acrylate, as is possible with the novel technique.
  • free-radical polymerization produces a crosslinked network that is generally less uniform than the networks that can be produced through step-grown polymerization of the thiol-acrylate.
  • Another advantage of the novel system is that little or no initiator decomposition products remain that might diffuse from the product into surrounding biological tissue.
  • hydroxyapatite can be added to the copolymerization mixture before or during curing to impart mechanical strength, and to provide osteogenic materials for promoting new bone growth.
  • a long-chain monomer such as a polyethylene glycol triacrylate or other multifunctional acrylate may be used to produce a product that is useful for soft tissue repair.
  • other naturally-occurring or synthetic molecules may be incorporated into the copolymer during the polymerization step, preferably by incorporating thiol-containing molecules into the polymerization mixture.
  • an antibody, peptide, enzyme, or protein with at least one free thiol group e.g., from a cysteine residue
  • a cysteine residue may be added to either end of a peptide or protein that may not otherwise have a suitable cysteine available for such a reaction.
  • a thiol-, alkene-, or alkyne-terminated oligonucleotide or nucleic acid sequence could be incorporated into the polymer during polymerization via the Michael addition mechanism.
  • FIGS. 1A-D depict the mechanism for a representative in situ, tertiary amine-catalyzed, anionic step growth polymerization in accordance with the present invention.
  • FIG. 2 depicts mass loss of various polymers after 7 days of incubation in stromal control media.
  • FIG. 3 depicts mass loss of various solid cast samples and foamed samples after 7 days of incubation in stromal control media.
  • the HA content was 20%.
  • FIG. 4 depicts the relative metabolic activity of hASC in thiol-acrylate extracts as measured by AlamarBlueTM fluorescent conversion.
  • the HA content was 20%.
  • FIG. 5 depicts relative metabolic activity of hASC exposed to 7 days of stromal control media extracts.
  • the HA content was 20%.
  • FIG. 6 depicts the compressive yield strength measured in mechanical tests of foamed and solid composites.
  • FIG. 7 depicts relative metabolic activity for hASC cultured on different solid composites.
  • the HA content was 20%.
  • FIG. 8 depicts relative metabolic activity for hASC cultured on foamed composites.
  • the HA content was 20%.
  • FIG. 9 depicts metabolic activity for hASC cultured on various scaffolds.
  • FIG. 10 depicts calcium deposition for cells cultured on various scaffolds in stromal and osteogenic media.
  • FIG. 11 depicts a quantitative measure of hASC proliferation on various scaffolds, using PicoGreenTM to determine total DNA content.
  • FIG. 12 depicts differences in the expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in hASC cultured on various scaffolds in stromal and osteogenic media.
  • the vertical axis depicts the observed fold-change.
  • FIGS. 13A-D depict SEM images of in vitro PETA-co-TMPMP foam and in situ PETA-co-TMPTMP foam.
  • FIGS. 14A-C depict micro-CT images of orthogonal slices of foamed scaffold samples.
  • compositions of this invention have broadly tunable mechanical and chemical properties.
  • Various biocompatible polymer and copolymer compositions containing thiol, and acrylate or alkyne moieties within the scope of the invention can be synthesized using the same general method and reaction scheme. By varying the number of functional moieties one may synthesize straight chain, branched, or cross-linked compositions.
  • gel times that are tunable across a wide range of times (typically, from minutes to hours).
  • the rate of reaction may be tuned by adjusting the concentration of in situ amine functionality.
  • the properties of the product may be tuned, for example, by modifying the degree of crosslinking, or by incorporating substituents into the thiol or acrylate comonomer.
  • an acrylate is the preferred co-monomer
  • alkynes may be used as the co-monomer as well.
  • the number of alkyne groups in the molecule may be varied from one to two to three or even more.
  • a mixture of acrylate and alkyne may be employed.
  • the stromal control media used in the prototype demonstrations comprised Dulbecco's Modified Eagle's Medium, 10% fetal bovine serum, and 1% triple antibiotic solution.
  • the osteogenic media used in the prototype demonstrations comprised Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, 1% triple antibiotic solution, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbate-2-phosphate, and 10 mM ⁇ -glycerophosphate.
  • compositions containing TMPTMP with di- or tri-functional acrylates were prepared in a 1:1 functionality ratio. These compositions were subjected to mass loss and hASC cytotoxicity tests (explained in Examples 4 and 5). Twenty stock solutions containing PETA, a preferred acrylate in terms of biocompatibility and mass loss data, and DEA (content ranging from 2.8-35.1%) were prepared, and the resulting copolymer compositions were subjected to mechanical testing (Example 6).
  • the strength of the materials can be altered by varying the initial DEA concentration.
  • the functionality and thus the cross-linking density are functions of the amine concentration.
  • the first Michael addition reaction with the secondary amine results in a loss of one acrylate functionality from the trifunctional acrylate.
  • a 16.1% DEA concentration was chosen for use in a preferred bone repair composition because it produced the highest Young's Modulus as measured in our initial experiments.
  • the composition had high elasticity and high mechanical strength.
  • a preferred gel time for many applications is on the order of 15-20 minutes, to allow time to mix and apply the material, while forming a material with suitable flexural strength within a practical time.
  • the gel time can be tuned by adjusting the concentration of amine, and the number of functional groups on the thiol or acrylate comonomer.
  • DEA and PETA were used to prepare a solution for both foamed and solid composite materials.
  • the DEA and PETA (16.1% DEA by acrylate molar functionality) were combined in advance, and stirred for 24 hours to form the in situ catalyst/comonomer.
  • TMPTMP was added in a 1:1 molar functionality ratio (i.e., the ratio of thiol groups to acrylate groups), and the material was mixed with a stir rod for 3 hours. Then several concentrations of HA (0%, 15%, 20%, 25% wt/wt) were added to the PETA-co-TMPTMP solution. This solution was cast into cylindrical molds (10 ⁇ 10 mm), and allowed to cure for 24 hours to form the solid composite copolymer material.
  • a foamed composite copolymer was prepared by pouring the PETA-co-TMPTMP with HA (0%, 15%, 20%, 25% wt/Wt) into a 250 mL pressurized spray canister using 7 g compressed nitrous oxide as a gas foaming agent. The mixture was expelled from the canister into the same types of cylindrical, polydimethylsiloxane molds as those used for the solid casting.
  • a PETA-co-TMPTMP+20% HA foamed sample was prepared in vitro by foaming directly into a beaker containing stromal control media (instead of the cylindrical molds) to observe the effect of physiological conditions on polymerization and foam structure. There was no inhibition of polymerization from the stromal aqueous media.
  • PCL+0% HA and PCL+20% HA foams were fabricated by thermally-induced phase separation from 1,4-dioxane, followed by lyophilization, as otherwise described in Zanetti A S, McCandless G T, Chan J Y, Gimble J M, Hayes DJ. Characterization of novel akermanite:poly - ⁇ - caprolactone scaffolds for human adipose - derived stem cells bone tissue engineering . Journal of Tissue Engineering and Regenerative Medicine Accepted Oct. 4, 2012.
  • the experimental groups were the following:
  • the mass of both foamed and solid composite polymers were measured.
  • the PCL foam polymers served as positive controls, and PETA-co-TMPTMP+0% HA served as an internal comparison. All samples were normalized versus the initial mass before media exposure. The samples were incubated on an orbital shaker with 5 mL stromal control media at 37° C., 200 rpm for 7 days. The samples were tested by the multiple headspace extraction method to determine mass loss. The resulting extracts were used in cytotoxicity testing.
  • the extracts from the mass loss test were filtered (0.22 ⁇ m pore size) and pipetted (100 ⁇ L/well) into a 96-well plate previously sub-cultured with hASC (2,500 cells/well).
  • the plates were placed in an incubator at 37° C. under 5% CO 2 for 24 hours.
  • the cellular viability on the scaffolds was determined by adding 10 ⁇ L AlamarBlue reagent to each well, and re-incubating at 37° C. under 5% CO 2 for 2 hours. Fluorescence was measured at an excitation wavelength of 530 nm and an emission wavelength of 595 nm using a fluorescence plate reader.
  • a tissue culture-treated plastic 96-well plate served as a control substrate.
  • Compression testing was performed on four specimens of each scaffold type with cylindrical geometry of 10 ⁇ 10 mm at room temperature using a hydraulic universal testing machine (Instron Model 5696, Canton, Mass., USA) at an extension rate of 0.5 mm/min, to a maximum compression strain of 90%.
  • the scaffolds tested were solid and foamed PETA-co-TMPTMP containing HA (0%, 15%, 20%, 25% wt/wt), and in vitro foamed PETA-co-TMPTMP+20% HA.
  • the measured compressive strengths of human cortical and cancellous bone are 130-180 MPa and 4-12 MPa, respectively.
  • Liposuction aspirates from subcutaneous adipose tissue were obtained from three healthy adult subjects (1 male, 2 female) undergoing elective procedures. All tissues were obtained with informed consent under a clinical protocol reviewed and approved by the Institutional Review Board at the Pennington Biomedical Research Center. Isolation of hASC was performed as otherwise described in Gimble J M, Guilak F, Bunnell BA. Clinical and preclinical translation of cell - based therapies using adipose tissue - derived cells . Stem Cell Research & Therapy 2010; 1.
  • Passage 2 of an individual cell culture was used for a cell viability test after acute exposure to the scaffold media extracts on loaded scaffolds in a spinner flask.
  • Passage 2 of an individual cell culture was used to evaluate in vitro hASC osteogenesis on tissue culture-treated polymers for different scaffold types.
  • PETA-co-TMPTMP copolymer The ability of the PETA-co-TMPTMP copolymer to support hASC adhesion and short-term culture was evaluated using AlamarBlue metabolic activity assays and examining cell morphology. (AlamarBlue is an indicator of cell viability and proliferation.)
  • the viability of cells within the scaffolds in stromal control media was determined after 7 days using an AlamarBlue metabolic activity assay.
  • the metabolic activity of cells within the scaffolds in stromal or osteogenic media was also determined using the AlamarBlue assay at 7, 14, and 21 days.
  • the scaffolds were removed from culture, washed three times in phosphate buffered saline, and incubated with 10% AlamarBlue in Hank's balanced salt solution without phenol red (pH 7) for 90 minutes.
  • Alizarin red staining which stains calcium-rich deposits, was used to assess the osteogenic potential of different scaffold types.
  • hASC osteogenesis (either for cells alone, or for cells with scaffolds) was assessed after 7, 14, and 21 days of culture in stromal or osteogenic media using alizarin red stain.
  • Cells and scaffolds were briefly washed four times with 0.9% NaCl (1 mL/well) and fixed with 70% ethanol (1 mL/well). The fixative was removed by aspiration; plates were stained with 2% alizarin red for 10 minutes, and then washed with deionized water six times.
  • Each scaffold type was imaged by SEM.
  • Solid PETA-co-TMPTMP copolymer samples were placed in a 12-well plate to form a thin layer (1 mm thick).
  • the polymers that had been seeded with stem cells were fixed for 30 minutes with 2% glutaraldhyde. Then all samples were dehydrated by washing with ethanol, starting with a 30% ethanol solution, increasing by 10% every 30 minutes to 100% ethanol. Overnight 100% hexamethyldisilazane was added to the samples to replace the dried air and ethanol.
  • An EMS550X sputter coater applied a conductive platinum coating for 2 minutes, followed by standard SEM imaging. SEM images were also taken of in situ and in vitro foamed samples.
  • Volume renderings were generated from three-dimensional data of foamed samples using Avizo Fire software, version 7.0.1 (Visualization Services Group). Two overlapping sub-volumes were rendered simultaneously, one with a red-orange-white color map corresponding to thiol-acrylate foam, and another with a blue-green color map corresponding to hydroxyapatite inclusions. Orthogonal slices were created using ImageJ, with equivalent scale, brightness, contrast, and grey map settings.
  • FIGS. 2 and 3 Mass loss after 7 days of incubation in stromal control media is illustrated in FIGS. 2 and 3 .
  • FIG. 2 illustrates, both PETA+0% HA and PETA+20% HA demonstrated greater stability than other experimental materials tested, with losses similar to those of the PCL control.
  • the composites of the present invention have greater physiological stability than that of previously reported composites, which is an advantage since bones typically take weeks or months to regenerate. TMPETA-containing polymers and composites degraded much more rapidly than those with PETA, and their stability correlated with the molecular weight of the oligomer.
  • FIG. 3 shows that the PETA+20% HA foam and solid had significantly greater mass loss than the PCL control foam. Without wishing to be bound by this hypothesis, the mass loss is believed to result from hydrolytic chain scission in a manner similar to the degradation of PCL in physiological solutions.
  • the PCL foam sample slightly increased in mass, perhaps from mineralization or non-specific protein deposition.
  • FIG. 4 shows the relative metabolic activity of hASC in thiol-acrylate extracts as measured by AlamarBlue fluorescent conversion. Relative fluorescent units are normalized versus live control. Asterisks indicate a sample that is significantly different from dead control. The conversion of AlamarBlue by PETA+0% HA and PETA+20% HA polymers was statistically not distinguishable from the tissue culture treated polymer and PCL control samples. It was also similar to the other materials tested.
  • Tissue culture-treated polystyrene served as the positive control
  • ethanol-treated hASC served as a negative control.
  • Cells exposed to both solid and foam PETA+0% HA and PETA+20% HA composite extracts had significantly higher metabolic activity than the dead control or cells exposed to the PCL foam extract ( FIG. 5 ).
  • the significant reduction in metabolic activity of hASC cultured with PCL extracts did not correlate with an increased reduction in mass of PCL scaffolds. This observation suggests that the extraction products from the novel copolymers should be less toxic than extracts from PCL products.
  • FIG. 6 shows measured compressive yield strengths in mechanical tests of foamed and solid PETA composites.
  • Human cancellous bone was the control, measured at 5.12 MPa.
  • the compressive strength of foamed PETA samples steadily increased with increasing HA content.
  • the solid samples behaved differently.
  • the addition of 15% HA resulted in a substantial increase in mechanical strength, but there was no significant change in yield strength for solid samples with higher concentrations of HA.
  • a foamed polymer will generally have lower mechanical strength than a solid polymer due to its porosity.
  • the porosity is presumably responsible for the differing trends seen in solid and foamed scaffolds as HA content increases. Increasing HA content reduced pore size, and the decreased pore volume resulted in a more solid-like and stronger structure in the foam samples. As porosity plays little or no role in the solid (non-foamed) samples, the HA content had less of an effect their mechanical strength.
  • Relative metabolic activity for hASC cultured on solid substrates is shown in FIG. 7 .
  • the results are normalized versus the live control. Asterisks indicate samples that were significantly different from live control.
  • the PETA samples and the PETA+HA samples were also significantly different from each other.
  • Cells were cultured on solid PETA composite samples for 7 days in stromal media and assayed for fluorescent AlamarBlue conversion; polystyrene treated tissue culture plates served as a positive control.
  • hASC cultured on both PETA scaffolds had significantly lower metabolic activity.
  • cells cultured on the PETA+20% HA composite had significantly lower metabolic activity than cells cultured on the PETA+0% HA sample.
  • this observation may reflect reduced the metabolic activity that is associated with the differentiation of stem cells exposed to HA, a known osteogenic compound; it does not necessarily reflect reduced biocompatibility.
  • FIG. 8 Relative metabolic activity for hASC cultured on foamed samples is shown in FIG. 8 .
  • the results are normalized versus the live control.
  • PETA+0% HA foam demonstrated higher metabolic activity than both the solid PETA+20% HA composite and the PCL+0% HA foam, but significantly lower metabolic activity than cells on tissue culture treated styrene (the live control).
  • the foamed PETA scaffold had a substantially larger surface area than that of the solid PETA scaffold, the results indicated that both forms of PETA supported hASC growth at levels similar to that of the PCL positive control.
  • the hASC were cultured under osteogenic and stromal control media for 21 days. Relative levels of AlamarBlue conversion, indicating metabolic activity, are shown in FIG. 9 (depicting relative intensity units).
  • PETA+0% HA showed highest metabolic activity, followed by PCL+0% HA. Almost no metabolic activity was measured in PETA+25% HA scaffolds, likely a result of smaller pore size and interconnectivity, reducing the number of viable cells within the scaffold.
  • HA was added either to PCL or PETA scaffolds, decreased metabolic activity was observed in stromal media but not in osteogenic media.
  • PCL control scaffolds with and without HA showed significant differences in metabolic activity at all time points.
  • the PETA+0% HA control also showed significant differences at 7 and 14 days.
  • PETA+15/20/25% HA samples showed no significant differences in metabolic activity between stromal and osteogenic conditions.
  • the reduced metabolic activity is common in osteogenic differentiation of mesenchymal stromal cells. Cells in osteogenic media would likely be differentiating regardless of the ceramic content of the scaffold, and therefore little or no difference in metabolic activity would be expected.
  • FIG. 10 illustrates calcium deposition on cells cultured on the PETA composites in stromal and osteogenic media, with PCL samples acting as controls.
  • alizarin red staining tended to be considerably higher for hASC cultured in osteogenic media as compared to samples cultured in stromal media.
  • the hASC cultured in osteogenic media showed a significant increase in staining with increased time in culture. Significant differences in calcium deposition were observed at 14 days between stromal and osteogenic media among all the scaffolds, except for PETA+25% HA. Both PETA+15% HA and PETA+20% HA showed increased staining at 21 days, significantly more than the PETA control. However, almost no calcium deposition was observed at 14 or 21 days for PETA+25% HA.
  • morphogenic proteins regulate osteogenesis.
  • the morphogenic proteins act on transcription factors (such as core binding factor alphal), and cause the activation of osteoblast-related genes such as those for alkaline phosphatase (ALP) and osteocalcin (OCN).
  • ALP alkaline phosphatase
  • OCN osteocalcin
  • the expression levels of these genes were used as markers for early- and middle-stage osteogenesis, respectively.
  • Quantitative reverse transcriptase polymerase chain reaction was used to assess the transcription of ALP mRNA at 7 days, and OCN at 14 and 21 days. The differences in the transcription levels of ALP and OCN on the varying scaffolds in stromal and osteogenic media are shown in FIG. 12 .
  • the cells on PETA+15% HA and PETA+20% HA scaffolds showed similar levels of transcription of ALP, and both were significantly higher than transcription on other PETA and PCL scaffolds.
  • Cells on PETA+0% HA scaffolds had higher ALP transcription than those on PCL+0% HA control scaffolds.
  • the OCN transcription in hASC as a function of scaffold type showed the same trend as ALP transcription, with cells cultured on PETA+15% HA and PETA+20% HA scaffolds demonstrating the greatest transcription of OCN.
  • Cells cultured on the PETA+25% HA sample exhibited low transcription of both markers, likely the result of poor cell proliferation in the absence of an interconnected, porous structure within the scaffold.
  • FIGS. 13A and 13C show SEM images of in vitro PETA+20% HA foam
  • FIGS. 13B and 13D show SEM images of in situ PETA+20% HA foam.
  • Scale bars are 100 ⁇ m in FIGS. 13A and 13B
  • 10 ⁇ m in FIGS. 13C and 13D SEM analysis indicated that there was no substantial difference in porosity or morphology between the in vitro and in situ foamed samples.
  • FIGS. 14A-C show micro-CT images of orthogonal slices of foamed scaffold samples analyzed using NIH ImageJ.
  • the scale bars are 500 ⁇ m.
  • the image data show good contrast between HA and polymer, confirming the suitability of micro-CT as an appropriate technique to image HA distribution and pore morphology.
  • Measurements using ImageJ from these datasets indicated pores ranging from 100-500 ⁇ m for the PETA+0% HA control, and 125-800 ⁇ m for the PETA+20% HA.
  • HA aggregations around 10-50 ⁇ m were seen; using a higher torque and a higher stirring speed may help improve homogeneity.
  • Three-dimensional micro-CT images (not shown) were used to visualize open cells and interconnectivity. These images showed that increasing the HA content resulted in smaller pore sizes and reduced porosity in the PETA composites. These effects were particularly evident at PETA+25% HA, for which the void spaces no longer appeared interconnected, and which had the appearance of a closed-cell foam. Increasing HA content above 25% did not appear to have much further effect on PCL void volume and interconnectivity. Because the PCL scaffold was synthesized by thermal precipitation, the pore size, volume, and interconnectivity are expected to be largely independent of solution viscosity. By contrast, increasing the HA content in PETA composite substantially affected viscosity, providing further support that the pore size and interconnectivity of the pore volume were affected by viscosity.
  • step growth nature of the amine-catalyzed Michael addition reaction used in this invention essentially eliminates the chance that unreacted monomer or free radicals might leaching from the scaffold, as could occur if instead a free-radical, chain-growth polymerization were used.
  • In situ polymerization permits absorbable foams to be used for conformal repairs of critical sized tissue defects, foams that can be easily delivered in a clinical or surgical setting.
  • novel foam represents a substantial improvement over prior PCL foams, which are formed externally prior to surgical insertion.
  • novel foams are also a substantial improvement over methyl methacrylate bone cements, which are largely inert, non-porous, and permanent.
  • the material is initially prepared as a two-component system, with the thiol and acrylate components in separate containers as part of an integrated delivery system.
  • the acrylate-containing portion contains both the in situ tertiary amine catalyst, and the acrylate monomer without amine.
  • the delivery system releases the thiol and acrylate components, mixes them in a static mixer, and injects the mixture under pressure into a tissue defect.
  • the copolymer then forms a foam and cures in situ to fill the defect site.
  • the thiol and acrylate components are contained separately in the barrels of a two-part syringe, fitted with a static mixing apparatus.
  • the plunger When the plunger is depressed, the thiol and acrylate materials are mixed and extruded into the tissue defect site where the copolymer can cure.
  • the thiol, acrylate, and amine in situ catalyst components are mixed in a container and poured or hand-filled into a defect site prior to curing.
  • the autoclaved stock/HA and TMPTMP/HA pre-polymer mixture was placed in a 250 mL pressurized spray canister using 7 g compressed nitrous oxide as a gas foaming agent.
  • the spray canister components were gas sterilized before surgical use.
  • the foamed composite copolymer was expelled from the canister into a sterilized pan.
  • the composite was cut into a rectangular prism with dimensions (15 mm ⁇ 10 mm ⁇ 1 mm) and placed into specimen (rat) 1.
  • specimens 2, 3, and 4 the pre-polymer mixture was prepared as described above, but was foamed into a 5 mL syringe instead of the sterile pan. 3 mL of foam from the syringe was injected on either side of the spinal column of a rat with previous bilateral decortication of the L4 and L5 transverse processes. The same process was used for specimen 5, except that the foam contained 0% HA.
  • the spinal column of specimen 3 was harvested at 3 weeks, while spinal columns from specimens 1, 2, 4, and 5 were harvested at 6 weeks.
  • specimens were treated with a subcutaneous injection of 0.5 mg/kg butorphanol (Torbugesic, Fort Dodge Animal Health) and 0.02 mg/kg glycopyrrolate (Robinul-V, Fort Dodge Animal Health, Fort Dodge, Iowa).
  • isoflurane 20 minutes later in an induction chamber.
  • the isoflurane was maintained at 1.5% via nose cone on a Bain circuit for the remainder of the procedure.
  • the lumbar region was clipped and aseptically prepared with 70% isopropanol/betadine.
  • a posterior midline skin incision was made over the lumbar spine.
  • Two fascial incisions were made 3 mm lateral and parallel to the spinous processes.
  • the L4 and L5 transverse processes were exposed using a combination of sharp and blunt dissection that was limited to the specific area of interest.
  • a scalpel was used to decorticate the transverse processes bilaterally.
  • the surgical sites were thoroughly lavaged with physiologic saline.
  • scaffolds were placed on both sides of the spine such that they spanned between the midpoint of each transverse process. Fascial and subcutaneous incisions were closed separately with 3-0 polyglactin 910 (Vicryl, Ethicon) in a simple continuous pattern. To inhibit migration, closure of the fascia around the implants effectively filled any potential space.
  • Subcutaneous tissue was apposed similarly. Tissue adhesive was used for skin closure. (Vetbond, 3M). The specimens were humanely euthanized by CO 2 asphyxiation 3 or 6 weeks after surgery.
  • the density of the defect site was tested using an x-ray line scanning method with Image J.
  • a ventral view of each spine was uploaded into Image J and analyzed with a density measuring tool.
  • a density line was drawn on the contralateral tissue region unaffected by the surgery or treatment, to act as a positive control.
  • the same density line was used to calculate the optical density (OD) of an area in the blank space of the image to serve as a negative control.
  • the density line was then moved to the defect site.
  • Using the same density line allowed a comparison of the optical density of the defect site with the positive and negative controls.
  • a ratio of each mean OD was used to normalize the percentage of bone growth in each specimen.
  • Each recorded density value represented the difference between the measured OD value and the OD for the blank space.
  • Specimen 3 (PETA:HA, 80:20, foamed in situ) was sacrificed three weeks post-operative. The spinal column containing the defect was removed. The spinal column was decalcified, and medial slices were cut and stained with Hematoxylin and eosin.
  • Specimen 1 pre-sculpted PETA:HA, 80:20
  • Specimen 2 PETA:HA, 80:20, in situ
  • Specimen 4 PETA:HA, 80:20, in situ
  • Specimen 5 PETA:HA, 100:0, in situ
  • Rats that were injected with the foamed thiol-acrylate nanocomposite showed some calcification at 3 weeks after implantation, which became more evident at 6 weeks after implantation (44.1% bone formation).
  • the increasing intensity of bone scaffolds indicated ossification of tissue intercalated into the scaffolds. (X-ray radiographs are not shown here, because they typically reproduce poorly in published patent documents.) Rats implanted with pre-molded samples, however, had a much lower increase in intensity at 6 weeks after implantation (37.1% bone formation).
  • Micro-CT was also performed to analyze bone formation. Avizo software was used to render micro-CT data. (The CT images are not shown here, because they typically reproduce poorly in published patent documents.) The CT images were generally consistent with the radiography results. Further, the rats injected with foamed in vivo scaffolds showed regions of discontinuous ossification in the scaffold area 6 weeks post-surgery. During the surgery, the structure of foamed in vivo sample may have been disrupted when the surgical site was closed. Reduced porosity and interconnectivity could then be the reason why new bone formation was seen to be non-continuous. Thus it is preferred that the surgical procedures used not be allowed to disrupt the foam; or that the foam be adequately cured before the surgical site is closed; or both.
  • Histology was analyzed for on the spinal defect from rat 3 (PETA:HA 80:20, foamed in situ) after sacrifice at 3 weeks. Decalcifying and staining the spinal column posed a practical problem in analyzing tissues for bone formation. For this reason, the structure of the tissues and the presence of osteoblasts were used to assess bone formation via histology. These images (also not shown here, because they typically reproduce poorly in published patent documents) showed early signs of cell organization and osteoblast formation. Rat 3 provided a comparison for assessing the other treatments. Lymphocytes were observed proximal to the implant at three weeks post-operative, but not in the rats six weeks post-operative.
  • Rat 1 pre-sculpted PETA:HA 80:20 was analyzed at six weeks. Significant signs of organized tissue were seen in comparison to Rat 3 at three weeks. The presence of osteoblasts in the tissue suggested bone growth at the defect site. (Bone per se was not seen due to the decalcification step employed.) Early signs of blood vessel formation were also seen, indicating that the implants were compatible with the surrounding tissue, and that they promoted tissue in-growth.

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